ABSTRACT The long-term goal of this research program is to elucidate the global mechanisms that coordinately regulate the expression of genes required for the development and differentiation of the ocular lens. Since the lens is a simple tissue composed of only two mature cell types, elucidation of these mechanisms provides insight into those processes required for the differentiation of far more complex tissues and provides the groundwork for the development and design of cutting-edge new avenues of biological research ranging from stem-cell replacement therapies to targeted cancer treatments. The central premise of this proposal is that differentiation of lens cells is dependent on the coordinated interactions of DNA- binding transcription factors with FGF and BMP signal transduction pathways to orchestrate lens-specific expression of hundreds of genes required to form the mature eye lens. The hallmark of mammalian lens fiber cell differentiation is accumulation of a- and b-/g-crystallins as key lens structural and protective proteins, cellular elongation, and degradation of nuclei and other organelles. In differentiating lens, specific groups of genes are transcriptionally turned on and off, and the central part of this process is controlled through the accessibility of chromatin DNA to associate with transcription factors and chromatin remodeling enzymes. Among these factors, c-Maf, Pax6, Prox1, c-Jun, Etv5, and Smads, act together as a unit to systematically regulate the spatial and temporal expression of individual crystallins and other genes essential for the differentiation and function of the eye lens. Specifically, this proposal will: 1) elucidate the functional role that chromatin plays in regulating lens differentiation-specific gene expression at the genome-wide level, (2) define the specific roles of DNA-binding transcription factors and their roles in formation of ?open? chromatin regions during lens differentiation, and (3) identify and characterize the role and function of distinct topologically associating domains (TADs) in lens cell nuclei, including transcriptional factories, nucleoli and splicing speckles, as critical components of the lens differentiation. The proposed studies are supported by strong preliminary data demonstrating the formation of ?open? regions of chromatin in the promoters and enhancers of key lens differentiation genes allowing accessibility and function of essential transcription factors and the identification of discrete TADs comprised of crystallin loci from different chromosomes to coordinate their expression in lens fiber cells. The results will define for the first time those sequential events required for lens-specific gene expression through the interplay between transcription factors and altered chromatin conformation, will provide novel insights into the 3D-organization of lens fiber cell nuclei that are required for lens differentiation, and uncover novel regulatory mechanisms that drive lens fiber cell denucleation.